The 18q syndrome is one of the most common nonlethal deletional defects in humans and is characterized by craniofacial anomalies, humoral deficiency and mental retardation. Although the deleted chromosomal region common to these patients is 18q2l.3 the genes associated with this phenotype have not been identified. The purpose of this study is to develop a large fragment cloning strategy which will help elucidate the molecular basis of a disorder which maps within approximately 5 megabases of uncharacterized chromosomal DNA. Yeast artificial chromosomes (YACs) are novel cloning vectors which permit the isolation of large regions of genomic DNA. However, the usefulness of YACs in the analysis of complex genomes was untested and depended upon the development of routine approaches to: 1) assess their fidelity in representing genomic structure, 2) locate and orient internal genes, 3) map restriction sites, and 4) rescue the terminal sequences-to provide probes used in isolating overlapping clones (chromosomal walking). Preliminary data suggests that these tasks can be accomplished. Further physical mapping and cloning of 18q2l.3 will open new avenues for assessing normal and pathologic gene regulation. Using DNA from 18q syndrome patients with interstitial deletions or translocations and the map as a reference, distinct breakpoints can be identified. Isolated breakpoints can be used to identify clones which should be examined for the presence of a new gene(s). Analysis of 18q2l.3 will help determine whether the variable phenotype is reflective of a contiguous gene syndrome or genomic imprinting. Finally, this approach may serve as a model for analyzing other chromosomal aberrations whose breakpoints reside in genomic regions inaccessible to conventional molecular cloning techniques.